Interaction of cell-cell adhesion molecules with the actin cytoskeleton is critical for the maintenance and stability of the glomerular filtration barrir formed by the slit diaphragm. Disrupting this interaction is intimately coupled to the initiation o proteinuria and progression of nephrotic syndromes in general. Many forms of glomerulosclerosis including focal and segmental glomerulosclerosis (FSGS) are characterized by reorganization of the actin cytoskeleton resulting in loss of cell-cell adhesion and displacement of the slit diaphragm. Most studies had focused on global regulation of the actin cytoskeleton without considering the possibility that perturbing actin assembly directly at sites o cell-cell adhesion might be the key to disrupting slit diaphragm stability. Recently, we found that FSGS1/?-actinin-4 is required for actin assembly at cell-cell contacts. Furthermore, a mutation of FSGS1/?-actinin-4 K255E that links to a dominant form of FSGS specifically inhibits actin assembly at sites of cell-cell adhesion without compromising the overall actin cytoskeletal organization. Therefore, disruption of actin assembly locally at adhesive contacts might be a convergent pathway that leads to break down of the slit diaphragm filtration barrier. Given the complexity of the slit diaphragm, dissecting the structural-functional relationships of its molecular components would be essential to revealing the mechanism behind development of proteinuria and FSGS. Aim 1. Determine how FSGS1/?-actinin-4 and its disease mutation regulate actin dynamics at membrane adhesion complex. We will use our newly developed reconstitution biochemical assays, in combination with actin reporter assays, to assess the role of FSGS1/?-actinin-4 in actin assembly. First, we will define the molecular function of the wild-type FSGS1/?-actinin-4. Second, we will analyze how to the disease mutant K255E inhibits actin assembly. Lastly, we will develop high throughput assays to screen for inhibitors of K255E mutation. Aim 2. Characterize FSGS1/?-actinin-4-associated actin regulatory machine. We developed a novel target-based crosslinking to identify six proteins in complex with FSGS1/?-actinin-4. We will dissect the molecular and cellular functions of these junctional components using in vitro biochemical and cellular approaches (shRNA and mutant expression) as well as high-resolution imaging techniques. Aim 3. Develop a novel pressure chamber apparatus to investigate the role of hydraulic pressure in the development of proteinuria. Actin assembly at cell-cell contacts is necessary for proper cell-cell adhesion to withstand normal and hypertensive pressure. We will use a new pressure chamber to perform functional analysis under different hydraulic pressure treatments to elucidate the causative role of disease susceptible genes in the initiation and progression of permeability barrier disruption.